LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the...LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the sequence was an open reading frame (ORF) of 735bp capable of encoding 245 amino acids. The polyhedrin gene sequences of the LdMNPV-NEFU isolate and a Canada strain, LdMNPV-G differed in 5 bases. The polyhedrin gene of the LdMNPV-NEFU isolate contained C, G, T, C and G at 54, 109,379, 508 and 701 sites from the start codon, but the LdMNPV-G isolate contained G, C, C, T and T at the corresponding sites respectively. The same amino acids were encoded by the two ORF sequences, with the exception that Asp and His are encoded by GAC on the polyhedrin gene sequence of the LdMNPV-NEFU isolate and by CAC in the LdMNPV-G isolate. The LdMNPV polyhedrin gene was expressed in E.coli BL21 (DE3) by the pT7-7 plasmid vector.展开更多
Baculoviruses are effective biological control agents for many insect pests. They not only efficiently challenge the host immune system but also make them hyperactive for better virus dispersal. Some investigations ha...Baculoviruses are effective biological control agents for many insect pests. They not only efficiently challenge the host immune system but also make them hyperactive for better virus dispersal. Some investigations have focused on the viral mechanisms for induction of such altered response from the host. However, there are no current studies monitoring changes in gene expression during this altered phenotype in infected larvae. The L. dispar multiple nucleopolyhedrovirus(Ld MNPV) induces hyperactivity in third instar L. dispar larvae at 3-days post infection(dpi), to continued till 6 dpi. The transcriptome profiles of the infected and uninfected larvae at these time points were analyzed to provide new clues on the response of the larvae towards infection during hyperactivity. Gene ontology enrichment analysis revealed, most of the differentially expressed genes(DEGs) were involved in proteolysis, extracellular region, and serine-type endopeptidase activity. Similarly, Kyoto Encyclopedia of Genes and Genome enrichment analysis showed maximum enrichment of 487 genes of the signal transduction category and neuroactive ligand–receptor interaction sub-category with 85 annotated genes. In addition, enrichment map visualization of gene set enrichment analysis showed the coordinated response of neuroactive ligand–receptor interaction genes with other functional gene sets, as an important signal transduction mechanism during the hyperactive stage. Interestingly all the DEGs in neuroactive ligand–receptor interactions were serine proteases, their differential expression during the hyperactive stage correlated with their conceivable involvement in disease progression and the resulting altered phenotype during this period. The outcome provides a basic understanding of L. dispar larval responses to Ld MNPV infection during the hyperactive stage and helps to determine the important host factors involved in this process.展开更多
American chestnut(Castanea dentata[Marsh.]Borkh.)was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen,Cryphonec-tria parasitica(Murrill)Barr,functionally eliminated it acros...American chestnut(Castanea dentata[Marsh.]Borkh.)was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen,Cryphonec-tria parasitica(Murrill)Barr,functionally eliminated it across its range.One promising approach toward restoring American chestnut to natural forests is development of blight-tolerant trees using genetic transformation.However,transformation and related processes can result in unexpected and unintended phenotypic changes,potentially altering ecologi-cal interactions.To assess unintended tritrophic impacts of transgenic American chestnut on plant herbivore interactions,gypsy moth(Lymantria dispar L.)caterpillars were fed leaf disks excised from two transgenic events,Darling 54 and Darling 58,and four control American chestnut lines.Leaf disks were previously treated with an LDso dose of either the speces-specific Lymantria dispar multiple nucleopolyhedrovirus(LdMNPV)or the generalist pathogen Bacillus thuringiensis subsp.kurstaki(Btk).Mortality was quantified and compared to water blank controls.Tree genotype had a strong efect on the efficacies of both pathogens.Larval mortality from Btk-treated foliage from only one transgenic event,Darling 54,differed from its isogenic progenitor,Ellis l,but was similar to an unre-lated wild-type American chestnut control.LdMNPV efficacy was unaffected by genetic transformation.Results suggest that although genetic modification of trees may affect interactions with other nontarget organisms,this may be due to insertion effects,and varia-tion amnon diMterent genvtypes(wlether uasgeic ul wild-typc)iupaults a giealci change in response than transgene presence.展开更多
文摘LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the sequence was an open reading frame (ORF) of 735bp capable of encoding 245 amino acids. The polyhedrin gene sequences of the LdMNPV-NEFU isolate and a Canada strain, LdMNPV-G differed in 5 bases. The polyhedrin gene of the LdMNPV-NEFU isolate contained C, G, T, C and G at 54, 109,379, 508 and 701 sites from the start codon, but the LdMNPV-G isolate contained G, C, C, T and T at the corresponding sites respectively. The same amino acids were encoded by the two ORF sequences, with the exception that Asp and His are encoded by GAC on the polyhedrin gene sequence of the LdMNPV-NEFU isolate and by CAC in the LdMNPV-G isolate. The LdMNPV polyhedrin gene was expressed in E.coli BL21 (DE3) by the pT7-7 plasmid vector.
基金supported by NSFC Grant(31670659)Special Fund for Forest Scientific Research in the Public Welfare(201404403-09)Shaanxi Provincial Science and Technology Innovation Project(2014KTCL02-14)
文摘Baculoviruses are effective biological control agents for many insect pests. They not only efficiently challenge the host immune system but also make them hyperactive for better virus dispersal. Some investigations have focused on the viral mechanisms for induction of such altered response from the host. However, there are no current studies monitoring changes in gene expression during this altered phenotype in infected larvae. The L. dispar multiple nucleopolyhedrovirus(Ld MNPV) induces hyperactivity in third instar L. dispar larvae at 3-days post infection(dpi), to continued till 6 dpi. The transcriptome profiles of the infected and uninfected larvae at these time points were analyzed to provide new clues on the response of the larvae towards infection during hyperactivity. Gene ontology enrichment analysis revealed, most of the differentially expressed genes(DEGs) were involved in proteolysis, extracellular region, and serine-type endopeptidase activity. Similarly, Kyoto Encyclopedia of Genes and Genome enrichment analysis showed maximum enrichment of 487 genes of the signal transduction category and neuroactive ligand–receptor interaction sub-category with 85 annotated genes. In addition, enrichment map visualization of gene set enrichment analysis showed the coordinated response of neuroactive ligand–receptor interaction genes with other functional gene sets, as an important signal transduction mechanism during the hyperactive stage. Interestingly all the DEGs in neuroactive ligand–receptor interactions were serine proteases, their differential expression during the hyperactive stage correlated with their conceivable involvement in disease progression and the resulting altered phenotype during this period. The outcome provides a basic understanding of L. dispar larval responses to Ld MNPV infection during the hyperactive stage and helps to determine the important host factors involved in this process.
基金Thanks to Hannah Nadel(USDA-APHIS)for kindly providing gypsy moth larvae,and John Podgwaite(USDA Forest Service)for providing purified LdM-NPV.We gratefully acknowledge Seaira Goetz and Elliot Hunsinger for their outstanding technical assistance in the laboratory.Substantive comments by T.Horton and M.K.Fierke improved earlier versions of the manuscript.This project was supported by Biotechnology Risk Assessment Grant Program competitive grant No.2012-33522-19863 from the USDA National Institute of Food and.Agriculture.
文摘American chestnut(Castanea dentata[Marsh.]Borkh.)was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen,Cryphonec-tria parasitica(Murrill)Barr,functionally eliminated it across its range.One promising approach toward restoring American chestnut to natural forests is development of blight-tolerant trees using genetic transformation.However,transformation and related processes can result in unexpected and unintended phenotypic changes,potentially altering ecologi-cal interactions.To assess unintended tritrophic impacts of transgenic American chestnut on plant herbivore interactions,gypsy moth(Lymantria dispar L.)caterpillars were fed leaf disks excised from two transgenic events,Darling 54 and Darling 58,and four control American chestnut lines.Leaf disks were previously treated with an LDso dose of either the speces-specific Lymantria dispar multiple nucleopolyhedrovirus(LdMNPV)or the generalist pathogen Bacillus thuringiensis subsp.kurstaki(Btk).Mortality was quantified and compared to water blank controls.Tree genotype had a strong efect on the efficacies of both pathogens.Larval mortality from Btk-treated foliage from only one transgenic event,Darling 54,differed from its isogenic progenitor,Ellis l,but was similar to an unre-lated wild-type American chestnut control.LdMNPV efficacy was unaffected by genetic transformation.Results suggest that although genetic modification of trees may affect interactions with other nontarget organisms,this may be due to insertion effects,and varia-tion amnon diMterent genvtypes(wlether uasgeic ul wild-typc)iupaults a giealci change in response than transgene presence.
